Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ0 cells).

To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ0 cells of human, mouse, and rat origin by polymerase chain reaction (PCR); (2) quantification of mtDNA by quantitative PCR (qPCR); (3) preparation of calibrator plasmids for mtDNA quantification; and (4) quantification of mtDNA by direct droplet digital PCR (dddPCR). © 2023 Wiley Periodicals LLC. Basic Protocol: Inducing mtDNA loss with mUNG1 Alternate Protocol 1: Generation of ρ0 cells by mtDNA depletion with EtBr and ddC Alternate Protocol 2: Generation of ρ0 cells by knocking out genes critical for mtDNA replication Support Protocol 1: Genotyping ρ0 cells by DirectPCR Support Protocol 2: Determination of mtDNA copy number by qPCR Support Protocol 3: Preparation of calibrator plasmid for qPCR Support Protocol 4: Determination of mtCN by direct droplet digital PCR (dddPCR).

The efficiency of the translesion synthesis across abasic sites by mitochondrial DNA polymerase is low in mitochondria of 3T3 cells.

Translesion synthesis by specialized DNA polymerases is an important strategy for mitigating DNA damage that cannot be otherwise repaired either due to the chemical nature of the lesion. Apurinic/Apyrimidinic (abasic, AP) sites represent a block to both transcription and replication, and are normally repaired by the base excision repair (BER) pathway. However, when the number of abasic sites exceeds BER capacity, mitochondrial DNA is targeted for degradation. Here, we used two uracil-N-glycosylase (UNG1) mutants, Y147A or N204D, to generate AP sites directly in the mtDNA of NIH3T3 cells in vivo at sites normally occupied by T or C residues, respectively, and to study repair of these lesions in their native context. We conclude that mitochondrial DNA polymerase γ (Pol γ) is capable of translesion synthesis across AP sites in mitochondria of the NIH3T3 cells, and obeys the A-rule. However, in our system, base excision repair (BER) and mtDNA degradation occur more frequently than translesion bypass of AP sites.

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions.

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.

Mitochondrial DNA ligase is dispensable for the viability of cultured cells but essential for mtDNA maintenance.

Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ(0) phenotype.

A retro-lentiviral system for doxycycline-inducible gene expression and gene knockdown in cells with limited proliferative capacity.

Currently, there is no reliable system for regulated gene expression and regulated gene knockdown in cells with finite lifespan. In this manuscript, we describe a vector system, consisting of a retrovirus for the delivery of rtTA, and a lentivirus for the delivery of either a transgene or a miR-shRNA for the modification of primary cells. Primary rat pulmonary microvascular endothelial cells (PMVEC) modified by these vectors for the inducible expression of Gaussia luciferase or DsRed Express demonstrated greater than 100-fold induction of the transgene expression with doxycycline. The system works reliably in both sequential and simultaneous infection modes, with about 95% of the sells selected with two antibiotics being inducible in each mode. The lentiviral vector for gene knockdown allows for the direct cloning of shRNA oligos using alpha-complementation, and for the monitoring of induction of RNA interference with fluorescent reporter, mCherry. The gene knockdown vector was validated by knocking down beta-actin expression in PMVECs, with two of the four constructs showing 59 and 75% knockdown, respectively, compared to uninduced controls. The vectors described here were successfully used for the modification of various primary and established cell lines for regulated gene expression and regulated knockdown.

West Nile virus genome amplification requires the functional activities of the proteasome.

The lifecycle of intracellular pathogens, especially viruses, is intimately tied to the macromolecular synthetic processes of their host cell. In the case of positive-stranded RNA viruses, the ability to translate and, thus, replicate their infecting genome is dependent upon hijacking host proteins. To identify proteins that participate in West Nile virus (WNV) replication, we tested the ability of siRNAs designed to knock-down the expression of a large subset of human genes to interfere with replication of WNV replicons. Here we report that multiple siRNAs for proteasome subunits interfered with WNV genome amplification. Specificity of the interference was shown by demonstrating that silencing proteasome subunits did not interfere with Venezuelan equine encephalitis virus replicons. Drugs that blocked proteasome activity were potent inhibitors of WNV genome amplification even if cells were treated 12 h after infection, indicating that the proteasome is required at a post-entry stage(s) of the WNV infection cycle.

Identification of mutated cyclization sequences that permit efficient replication of West Nile virus genomes: use in safer propagation of a novel vaccine candidate.

Existing live-attenuated flavivirus vaccines (LAV) could be improved by reducing their potential to recombine with naturally circulating viruses in the field. Since the highly conserved cyclization sequences (CS) found in the termini of flavivirus genomes must be complementary to each other to support genome replication, we set out to identify paired mutant CS that could support the efficient replication of LAV but would be unable to support replication in recombinant viruses harboring one wild-type (WT) CS. By systematic evaluation of paired mutated CS encoded in West Nile virus (WNV) replicons, we identified variants having single and double mutations in the 5'- and 3'-CS components that could support genome replication at WT levels. Replicons containing only the double-mutated CS in the 5' or the 3' ends of the genome were incapable of replication, indicating that mutated CS could be useful for constructing safer LAV. Despite the identity of the central portion of the CS in all mosquito-borne flaviviruses, viruses carrying complementary the double mutations in both the 5'- and the 3'-CS were indistinguishable from WT WNV in their replication in insect and mammalian cell lines. In addition to the utility of our novel CS pair in constructing safer LAV, we demonstrated that introduction of these mutated CS into one component of a recently described two-component genome system (A. V. Shustov, P. W. Mason, and I. Frolov, J. Virol. 81:11737-11748, 2007) enabled us to engineer a safer single-cycle WNV vaccine candidate with reduced potential for recombination during its propagation.

Construction and characterization of a second-generation pseudoinfectious West Nile virus vaccine propagated using a new cultivation system.

Safer vaccines are needed to prevent flavivirus diseases. To help develop these products we have produced a pseudoinfectious West Nile virus (WNV) lacking a functional C gene which we have named RepliVAX WN. Here we demonstrate that RepliVAX WN can be safely propagated at high titer in BHK cells and vaccine-certified Vero cells engineered to stably express the C protein needed to trans-complement RepliVAX WN growth. Using these BHK cells we selected a better growing mutant RepliVAX WN population and used this to generate a second-generation RepliVAX WN (RepliVAX WN.2). RepliVAX WN.2 grown in these C-expressing cell lines safely elicit strong protective immunity against WNV disease in mice and hamsters. Taken together, these results indicate the clinical utility of RepliVAX WN.2 as a vaccine candidate against West Nile encephalitis.

Early production of type I interferon during West Nile virus infection: role for lymphoid tissues in IRF3-independent interferon production.

Infection of cells with flaviviruses in vitro is reduced by pretreatment with small amounts of type I interferon (IFN-alpha/beta). Similarly, pretreatment of animals with IFN and experiments using mice defective in IFN signaling have indicated a role for IFN in controlling flavivirus disease in vivo. These data, along with findings that flavivirus-infected cells block IFN signaling, suggest that flavivirus infection can trigger an IFN response. To investigate IFN gene induction by the very first cells infected during in vivo infection with the flavivirus West Nile virus (WNV), we infected mice with high-titer preparations of WNV virus-like particles (VLPs), which initiate viral genome replication in cells but fail to spread. These studies demonstrated a brisk production of IFN in vivo, with peak levels of over 1,000 units/ml detected in sera between 8 and 24 h after inoculation by either the intraperitoneal or footpad route. The IFN response was dependent on genome replication, and WNV genomes and WNV antigen-positive cells were readily detected in the popliteal lymph nodes (pLN) of VLP-inoculated mice. High levels of IFN mRNA transcripts and functional IFN were also produced in VLP-inoculated IFN regulatory factor 3 null (IRF3(-/-)) mice, indicating that IFN production was independent of the IRF3 pathways to IFN gene transcription, consistent with the IFN type produced (predominantly alpha).

Mutations in West Nile virus nonstructural proteins that facilitate replicon persistence in vitro attenuate virus replication in vitro and in vivo.

West Nile virus (WNV) infections in vertebrates are generally acute but persistent infections have been observed. To investigate the ability of WNV to produce persistent infections, we forced subgenomic WNV replicons to replicate within a cell without causing cell death. Detailed analyses of these cell-adapted genomes revealed mutations within the nonstructural protein genes NS2A (D73H, M108K), NS3 (117Kins), NS4B (E249G) and NS5 (P528H). WNV replicons and WNVs harboring a subset of NS2A or NS3 mutations showed a reduction in genome replication, a reduction in antigen accumulation, a decrease in cytopathic effect, an increased ability to persist in cell culture and/or attenuation in vivo. Taken together, these data indicate that WNV with a defect in replication and an increased potential to persist within the host cell can be generated by point mutations at multiple independent loci, suggesting that persistent viruses could arise in nature.

Evaluation of replicative capacity and genetic stability of West Nile virus replicons using highly efficient packaging cell lines.

A stable cell system for high-efficiency packaging of West Nile virus (WNV) subgenomic replicons into virus-like particles (VLPs) was developed. VLPs could be propagated on these packaging cells and produced infectious foci similar to foci produced by WNV. Focus size correlated with the replicative capacity of WNV replicons, indicating that genome copy number, rather than amount of trans-complementing structural proteins, was rate-limiting in packaging of VLPs. Comparison of VLP production from replicon genomes encoding partial or complete C genes indicated that portions of C downstream of the cyclization sequence could improve genome replication or that cis expression of C could enhance packaging. Interestingly, a rapid loss of replicon-encoded reporter gene activity was detected within two serial passages of reporter gene-containing VLPs. The loss of reporter activity correlated with gene deletion and better VLP growth, indicating a powerful selection pressure for WNV genomes lacking reporter genes.

Replication and clearance of Venezuelan equine encephalitis virus from the brains of animals vaccinated with chimeric SIN/VEE viruses.

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.

Sindbis virus with a tricomponent genome.

We established a system for propagation of Sindbis virus (SIN)-based replicons in tissue culture in the form of a tricomponent genome virus. Three RNA fragments containing complementing genetic information required for virus replication are packaged into separate viral particles, and each cell produces at least 1,000 packaged replicons and the number of packaged helpers sufficient to perform the next passage. This system can be used to generate large stocks of packaged replicons. The formation of infectious recombinant SIN virus was not detected in any experiments. These features make multicomponent genome SIN an attractive system for a variety of research and biotechnology applications.

Changes of the secondary structure of the 5' end of the Sindbis virus genome inhibit virus growth in mosquito cells and lead to accumulation of adaptive mutations.

Both the 5' end of the Sindbis virus (SIN) genome and its complement in the 3' end of the minus-strand RNA synthesized during virus replication serve as parts of the promoters recognized by the enzymes that comprise the replication complex (RdRp). In addition to the 5' untranslated region (UTR), which was shown to be critical for the initiation of replication, another 5' sequence element, the 51-nucleotide (nt) conserved sequence element (CSE), was postulated to be important for virus replication. It is located in the nsP1-encoding sequence and is highly conserved among all members of the Alphavirus genus. Studies with viruses containing clustered mutations in this sequence demonstrated that this RNA element is dispensable for SIN replication in cells of vertebrate origin, but its integrity can enhance the replication of SIN-specific RNAs. However, we showed that the same mutations had a deleterious effect on virus replication in mosquito cells. SIN with a mutated 51-nt CSE rapidly accumulated adaptive mutations in the nonstructural proteins nsP2 and nsP3 and the 5' UTR. These mutations functioned synergistically in a cell-specific manner and had a stimulatory effect only on the replication of viruses with a mutated 51-nt CSE. Taken together, the results suggest the complex nature of interactions between nsP2, nsP3, the 5' UTR, and host-specific protein factors binding to the 51-nt CSE and involved in RdRp formation. The data also demonstrate an outstanding potential of alphaviruses for adaptation. Within one passage, SIN can adapt to replication in cells of a vertebrate or invertebrate origin.

Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic.

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.

Roles of nonstructural protein nsP2 and Alpha/Beta interferons in determining the outcome of Sindbis virus infection.

Alphaviruses productively infect a variety of vertebrate and insect cell lines. In vertebrate cells, Sindbis virus redirects cellular processes to meet the needs of virus propagation. At the same time, cells respond to virus replication by downregulating virus growth and preventing dissemination of the infection. The balance between these two mechanisms determines the outcome of infection at the cellular and organismal levels. In this report, we demonstrate that a viral nonstructural protein, nsP2, is a significant regulator of Sindbis virus-host cell interactions. This protein not only is a component of the replicative enzyme complex required for replication and transcription of viral RNAs but also plays a role in suppressing the antiviral response in Sindbis virus-infected cells. nsP2 most likely acts by decreasing interferon (IFN) production and minimizing virus visibility. Infection of murine cells with Sindbis virus expressing a mutant nsP2 leads to higher levels of IFN secretion and the activation of 170 cellular genes that are induced by IFN and/or virus replication. Secreted IFN protects naive cells against Sindbis virus infection and also stops viral replication in productively infected cells. Mutations in nsP2 can also attenuate Sindbis virus cytopathogenicity. Such mutants can persist in mammalian cells with defects in the alpha/beta IFN (IFN-alpha/beta) system or when IFN activity is neutralized by anti-IFN-alpha/beta antibodies. These findings provide new insight into the alphavirus-host cell interaction and have implications for the development of improved alphavirus expression systems with better antigen-presenting potential.